Nearly 10% of the US population currently suffers from asthma. Evidence suggests that early life microbial exposure, both commensal and pathogenic, influences long-term risks for the development and severity of asthma. Studies of asthma in children suggest a role for nontypeable H. influenzae bacteria (NTHi) in the development of disease, as NTHi colonization of the nasopharynx in the first 4 weeks of life is associated with an increased risk of developing asthma by age 5 years. This finding suggests that NTHi infection during a specific window of susceptibility might influence programming of the immune system and establish a trajectory towards a more inflammatory outcome. Thus, prevention of early NTHi colonization might reduce the incidence of childhood asthma. Conversely, there are also data suggesting that early life exposure to specific antigens such as dog and cat dander is correlated with reduced risk of developing asthma later in life. The identification of both early life sensitizing and protective factors against the development of juvenile asthma is the major long-term goal of our research. Our preliminary data demonstrate that 3-day-old mice intranasally colonized with NTHi were significantly more likely to have exacerbated allergic airways disease in response to juvenile ovalbumin allergen exposure when compared to mice that were treated with saline alone as neonates. We hypothesize that asthma susceptibility is established in an early life window of competitive sensitizing and protective exposures that include NTHi in the neonatal respiratory tract and environmental antigens such ass dog and cat dander. We predict that neonatal NTHi exposure alters a critical immune checkpoint and establishes a trajectory towards juvenile immune and airway hyper-responsiveness upon exposure to common environmental allergens. Aim 1 will compare the immune and airway responses between animals with and without neonatal colonization with NTHi and juvenile sensitization to the major environmental allergens Aspergillus and dust mite Der p 1. Experiments will subsequently identify the window of susceptibility to NTHi immune reprogramming and identify associated post-colonization lung and peripheral blood mononuclear cell immune signatures that predict enhanced juvenile immune responsiveness and airway hyper-responsiveness to the allergen exposures. Aim 2 will determine how cat and dog antigens alter the early life immune checkpoint and juvenile airway hyperreactivity that follows neonatal NTHi colonization. The results of our proposed studies will contribute novel insights into how early life microbial and environmental exposures affect immune set points for childhood and adult disease like asthma.